[Microbiologia Medica 2016; 31:6103] [page 63] August Compte, a French philosopher of science, back in the
18th Century wrote: You do not know a science, if you do not know its history.
It is ambitious to adapt this aphorism to the history of the micro-biological diagnosis of Chlamydia trachomatis infection, but the father of positivism would probably forgive me…
In 1907, in Java, Ludwig Halberstaeder and Stanislaus von Prowazec described − using a light microscope − the presence of intracytoplasmatic inclusions in samples of conjunctival scrapings obtained from an orangutan that was experimentally infected with ocular material from a patient suffering from trachoma.
They made this observation by using the differential staining method patented three years before by Gustav Giemsa (10).
These scientists described not only the large intracytoplasmatic inclusions, but also some small extracellular particles. This was the first time that these microorganisms were classified as Chlamidozoa. It is well know that these microbes were for a long time considered as viruses.
Nowadays, Chlamydia spp. are classified as obligate intracellular bacteria that must parasitize eukaryotic cells to achieve a full developmental cycle. C. trachomatis shows a particular tropism for the genital and conjunctival epithelia and encompasses 19 differ-ent serovars, which are predominantly pathogenic for the urogen-ital tract (6).
Staining methods
Staining Chlamydiae with the Gram method gives extremely poor results for two main reasons: i) these microbes are growing in an intracellular site and ii) any muramic acid is lacking in their cell wall. Although the direct detection of Chlamydia in appropriate cyto-logical smears was performed by Gupta in 1979 (9), the diagnostic value of the method based on direct smear was questioned and not approved (15).
As an alternative technique, the iodine stain is much simpler for the detection of Chlamydia related intracytoplasmatic inclusions in infected cells, based on the large production of iodine stainable glycogen by C. trachomatis (19).
Culture
Back in 1932, the so-called lymphogranuloma venereum (LGV) agent was first cultivated in Guinea pigs’ kidney and testicles (16). The culture of clinical samples on cells monolayer (largely used the McCoy line, derived from mouse connective tissue) was for a long-time the gold standard (8) in Chlamydia diagnostics. The character-istic chlamydial inclusion body can also (and likely with a higher specificity) be detected after staining with fluorescence conjugated specific (either polyclonal but preferably monoclonal) antibodies that bind to different targets, including the chlamydial major membrane protein (MOMP) or the LPS. The sensitivity of culture-based tech-niques varies from 50% to 90% (3) depending on selected settings.
Culture is nowadays considered a slow, labour-intensive and hence quite costly method and it is very seldom used for routine diagnostics. Nevertheless, it retains an excellent level of specificity and allows the in vitro antibiotic susceptibility testing and the determination of serovar specificity of isolated strains, which may be advantageous in epidemiological studies.
At the end of the 1980s, two new diagnostic approaches become available for the detection of Chlamydia spp. specific antigens in the clinical microbiology laboratory: the enzyme linked immunoas-says (EIA) and the direct detection by immunofluorescence (DFA).
Enzyme linked immunoassays
This method allows the direct antigen detection based on the well-known enzyme-linked immunoassay technique (12). The average sensitivity of EIAs is lower, compared with the cell culture procedure, but their negative predictive value is high. The EIA-based methods can be performed in a single test format, but more Correspondence: Enrico Magliano, AMCLI, Via Farini 81, 20159 Milano (MI).
E-mail: e.magliano@libero.it
Key words: Chlamydia trachomatis infection; history.
Conflict of interest: the authors declare no potential conflict of interest. Received for publication: 21 June 2016.
Accepted for publication: 21 June 2016.
©Copyright E. Magliano, 2016 Licensee PAGEPress, Italy Microbiologia Medica 2016; 31:6103 doi:10.4081/mm.2016.6103
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 4.0) which permits any noncom-mercial use, distribution, and reproduction in any medium, provided the orig-inal author(s) and source are credited.
Journal of Entomological and Acarological Research 2012; volume 44:e
Microbiologia Medica; volume 31:6103
The microbiological diagnosis of
Chlamydia trachomatis
infections:
milestones from a centenary history
Enrico Magliano
Scientific Director, Italian Association of Clinical Microbiologists, Milan, Italy
Non-commercial
[page 64] [Microbiologia Medica 2016; 31:6103] frequently these techniques are fully automated with a
software-based interpretation of results.
In Italy, in 1988, an EIA-based national wide-large epidemiologi-cal study about the urogenital Chlamydia infections in patients attending specialized department of Roma, Milano and Torino was performed and this is still one of the most comprehensive study ever done in Italy in the field of sexually transmitted infections (STI) (24).
Direct detection by immunofluorescence
The direct fluorescent antibody test has high sensitivity in par-ticular when cervical specimens are tested.
The method is somehow complicated since the requirement of highly trained and specialized operators is required in order to keep high level of clinical performance and it is not suitable for process-ing large numbers of sample (17) simultaneously.
Serology based techniques
Until 1936, the complement fixation test, devised by S.B Bedson, Professor of Bacteriology at the London Hospital (1), was the only available method for the diagnostic detection of the antibody response. Among serological techniques, the Bedsonian comple-ment fixation test has been a time-honoured method, but subse-quently it was shown to have limited clinical diagnostic value. When applied to the issue of Chlamydia infections, a cross reactiv-ity was clearly observed among cases of psittacosis and oculo geni-tal infections, likely due to the wide cross reactivity of the antigens used (2). In 1962, Nichols and Mc Comb suggested the use of fluo-rescent-labelled antibodies method for the serological differentia-tion of Chlamydia spp. (18).
Later on, in 1970, Wang and Grayston (26) developed a new indi-rect micro-titre immunofluorescence typing test, which confirmed the existence of six major chlamydia serotypes involved in oculo-genital infections. In 1975, the same Authors identified an addi-tional group of 15 more serotypes. In 1983, Saikku e Paavonen (22) developed the first solid-phase enzyme assay (EIA) for detection of Chlamydia specific antibodies.
The use of serological test for diagnosis of uncomplicated C. tra-chomatis infections, due to the low sensitivity and specificity of the method has a very limited clinical value. As the 2016 European Guideline for the management of NGU suggest (13), the serologic data have only a limited clinical value and could be, if interpreted with caution, of some aid in the diagnosis of PID, ectopic pregnan-cy, reactive arthritis, neonatal pneumonia and tubal infertility.
Nucleic acid amplification test
The first experimental molecular characterization of Chlamydia were performed at the end of the last century’s 80s (20) followed by the development of many different molecular diagnostic based methods for the practical clinical use (23).
Actually, the diagnostic reference methods are based on NAATs (Nucleic Acid Amplification Test) technology that has intrinsic high specificity and sensitivity. By using NAATs the detectability of chlamydial infections has increased by some 50% compared to the previous methods (25).
Moreover, by using NAATs, it is possible to obtain the diagnosis with the usage of self-collectable and non-invasively obtained clin-ical samples such as first void urine or self-taken vaginal swabs. Today only NAATs are recommended by National and International Reference Centres and by international Guide lines (5, 13).
The POC (Point of Care) tests commonly based on antigen mem-brane capture on lateral flow assays are simple to perform, but these methods have low and clinically not adequate sensitivity so that their overall performance is alarming poor (14).
The Clinical Microbiologist, who participates to the on-going rev-olutionary evolution of the diagnostics tools, must be, at the same time, well informed and aware about the social and behaviour mod-ifications that have developed since the last century. In particular, it is of fundamental relevance to have a deep knowledge of all the novel sexual risk behaviours, especially among the most vulnerable communities, that have generated an increased risk for the acqui-sition and spreading of STI. Only being a major player in the game, the Clinical Microbiologists could be effective in the implementa-tion of effective preventive measures.
The recent Editorial published in the British Medical Journal (4) on the Chem sex underlined that violent, traumatic prolonged (days) sexual intercourse may facilitate the bacterial invasion of the GU tract. Again, recently, the Lymphogranuloma venereum has been reported as a re-appearing infection in vulnerable drug addict-ed patients in selectaddict-ed communities. It is so highly probable that the facilitated bacterial invasion is increased following violent and prolonged intercourse: and this will be a major issue also in Italy.
The 2016 Report of the EDC on the guidance for the control of Chlamydia infection in Europe (7 march 2016) confirmed that Chlamydia related disease are the most frequently reported sexual transmitted infection in the EU member States and in the neigh-bouring Countries (11). As a consequence, the Public Health System in England published in March 2016 a Report of the National Chlamydia Screening Program (21). This report has gen-erated a set of estimates about the epidemiology of chlamydia infections, from its incidence, prevalence and duration of infection, and its role in the pathogenesis of PID and tubal factor infertility (TFI). This report clearly supported the necessity to implement a nationwide screening of prevalent cases to prevent PID cases and suggested that greater emphasis should be placed on the detection and treatment of incident C. trachomatis infections.
Data have also been confirmed in our country by a collaborative joint effort between the Istituto Superiore di Sanità (Centro Operativo AIDS) and the AMCLI Study group on STI (GLIST).
Another stimulating field of interest are the studies performed on pathogenesis of the genital tract diseases due to C. trachomatis (7). Although the pathologic consequences of C. trachomatis geni-tal infections are well established, the mechanisms that result in the chlamydia induced tissue damage are not fully understood. Abundant in vitro data suggest that inflammatory response to chlamydia is initiated and sustained by actively infected non-immune host epithelial cells. Continued studies about the complex molecular and cellular interactions between these pathogen and the complex system of the human GU tract are needed to address the still relevant gaps in our understanding of these mechanisms.
At the end of this quite concise synthesis of the long history of the relationship between the clinical microbiologists and some of the most intriguing germs, such as chlamydiae, it is obvious to me to recall when during the International Meeting on Chlamydia that AMCLI organized in Como (21-22 may 2009) I asked my Colleague David Taylor Robinson, one of the most expert and prominent
Editorial
Non-commercial
tist that dedicated almost his entire life to the study of STI: Which is the role of the Clinical Microbiologist in the investigation of chlamydia related infections?. Well, his trenchant answer was Just fundamental.
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